The Bacillus cereus sensu lato (Bcsl) group includes B. anthracis, B. thuringiensis, and B. cereus; B. anthracis is the most pathogenic for humans. B. cereus strains typically cause foodborne illness in humans, though B. cereus strains have been identified as the etiological agents of a potentially fatal anthrax-like respiratory disease. Horizontal transfer of mobile genetic elements, such as plasmids and pathogenicity islands, is common among Bcsl group organisms. B. cereus G9241 (Bc9241), which was isolated from a welder in Texas who survived an anthrax-like respiratory illness, produces the anthrax lethal toxin (LT) and edema toxin (ET) as well as two polysaccharide capsules. Bioinformatics studies predicted that Bc9241 encodes a previously uncharacterized toxin Certhrax with ADP-ribosyltransferase (ADP-r) activity. We demonstrated that Certhrax is an ADP-r toxin that stimulates actin reorganization to cause cytotoxicity in cultured human epithelial cells. We also showed that Certhrax ADP-ribosylates vinculin (Vin) as its sole intracellular host target. In addition, Certhrax may enhance the action of LT since decreased actin association with the Anthrax Toxin Receptor-1 (ANTXR-1) increases the affinity of the anthrax toxin binding subunit Protective Antigen (PA) for ANTXR-1. The studies proposed in this application will address the molecular mechanism by which Certhrax evokes cytotoxicity and the potential role of the toxin in Bc9241 pathogenesis. Our hypothesis is that Certhrax ADP-ribosylates Vin at the host cell membrane so that Vin's interaction with focal adhesion proteins such as talin is disrupted. The subsequent release of ADP-ribosylated-Vin from the membrane disrupts downstream signaling which leads to cell death. We further speculate that Certhrax contributes to Bc9241 virulence by mechanisms that may include lowering the dose of LT needed to intoxicate macrophages, cells targeted by LT during B. anthracis infection. Our specific aims are to: (1) characterize ADP-ribosylation of Vin by Certhrax: to determine the mechanism by which the ADP-ribosylation disrupts the actin cytoskeleton to cause cell death, and ascertain if and how Certhrax influences protective antigen binding to the eukaryotic anthrax toxin receptor: and (2) assess the role of holo-Certhrax in macrophage intoxication and in Bc9241 virulence in established mouse models of B. cereus infection. In addition to the capacity to cause lethal disease in some hosts, Bc9241 is a potential reservoir for virulence factors like the anthrax toxins, which could be transferred horizontally to other Bacillus spp. and/or other Gram- positive pathogens. Completion of these studies will advance our understanding of the molecular action of Certhrax and will define how Certhrax contributes to the pathogenesis of Bc9241 disease. Our previous studies on Certhrax form the foundation of this application in which cell-based assays will be coupled with in vivo experimentation to dissect a heretofore unrecognized pathogenic strategy (ADP-ribosylation of host Vin) employed by a newly recognized pathogen with lethal infectious potential for humans.